Methods and compositions for the inhibition of Pin1

ABSTRACT

The invention features compositions and methods for inhibiting the Pin1 protein, and the treatment of disorders characterized by elevated Pin1 levels.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.61/656,806, filed Jun. 7, 2012, which is hereby incorporated byreference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grants DA031663and CA167677 awarded by NIH numbers NIH R03DA031663 and R01CA167677. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

In general, the invention relates to compositions and methods forinhibiting Pin1 and the treatment of disorders characterized by elevatedPin1 levels (e.g., immune disorders and proliferative disorders) withcompounds defined herein.

BACKGROUND OF THE INVENTION

Immune disorders are characterized by the inappropriate activation ofthe body's immune defenses. Rather than targeting infectious invaders,the immune response targets and damages the body's own tissues ortransplanted tissues. The tissue targeted by the immune system varieswith the disorder. For example, in multiple sclerosis, the immuneresponse is directed against the neuronal tissue, while in Crohn'sdisease the digestive tract is targeted.

Immune disorders affect millions of individuals and include conditionssuch as asthma, allergic intraocular inflammatory diseases, arthritis,atopic dermatitis, atopic eczema, diabetes, hemolytic anaemia,inflammatory dermatoses, inflammatory bowel or gastrointestinaldisorders (e.g., Crohn's disease and ulcerative colitis), multiplesclerosis, myasthenia gravis, pruritis/inflammation, psoriasis,rheumatoid arthritis, cirrhosis, and systemic lupus erythematosus.

Current treatment regimens for immune disorders typically rely onimmunosuppressive agents. The effectiveness of these agents can vary andtheir use is often accompanied by adverse side effects. Thus, improvedtherapeutic agents and methods for the treatment of autoimmune disordersare needed.

Additionally, the increased number of cancer cases reported in theUnited States, and, indeed, around the world, is a major concern.Currently there are only a handful of detection and treatment methodsavailable for some specific types of cancer, and these provide noabsolute guarantee of success. In order to be most effective, thesetreatments require not only an early detection of the malignancy, but areliable assessment of the severity of the malignancy.

It is apparent that the complex process of tumor development and growthmust involve multiple gene products. It is therefore important to definethe role of specific genes involved in tumor development and growth andidentify those genes and gene products that can serve as targets for thediagnosis, prevention, and treatment of cancers.

We and others have shown that Pin1 is prevalently overexpressed in humancancers and that high Pin1 marker levels correlate with poor clinicaloutcome in many cancers. In contrast, the Pin1 polymorphism that reducesPin1 expression is associated with reduced cancer risk in humans.Significantly, Pin1 activates at least 19 oncogenes/growth enhancers,including β-catenin, cyclin D1, NF-κB, c-Jun, c-fos, AKT, A1B1,HER2/Neu, MCl-1, Notch, Raf-1, Stat3, c-Myb, Hbx, Tax, and v-rel, andalso inactivates at least 12 tumor suppressors/growth inhibitors,including PML, SMRT, FOXOs, RARa, and Smad. Whereas Pin1 overexpressioncauses cell transformation and tumorigenesis, Pin1 knockdown inhibitscancer cell growth in cell cultures and mice. Pin1-null mice are highlyresistant to tumorigenesis induced either by oncogenes such as activatedRas or HER2/Neu, or tumor suppressors such as p53. Thus, there is a needin the art for Pin1 inhibitors to suppress numerous oncogenic pathwayssimultaneously for treating aggressive and/or drug-resistant cancers.

SUMMARY OF THE INVENTION

The current invention features compositions and methods for inhibitingPin1 by contacting the Pin1 protein with a Table 1 Compound. The Pin1protein can be within a cell, e.g., a human cell, such as a diseasedhuman cell. Table 1 Compounds can be administered in a therapeuticallyeffective amount for treating a subject, e.g., human subject, sufferingfrom or at risk of an immune disorder or a proliferative disorder.

In one aspect of the invention, the expression level of Pin1 marker in asubject maybe determined prior to administering a Table 1 Compound. Theexpression level of Pin1 marker level can be determined by collecting asample, e.g., a tissue sample, such as a blood or biopsy sample, fromthe subject and analyzing the expression of the Pin1 marker usingmethods known in the art. A Table 1 Compound can be administered if theexpression level of the Pin1 marker is elevated in the subject. Thesubject can have elevated Pin1 expression levels and can be sufferingfrom an immune disease or a proliferative disease.

In another aspect of the invention, the expression level of Pin1 markercan be determined after administration of a Table 1 Compound fordetermining efficacy of treatment and disease prognosis. Elevated Pin1marker level can be due to an inherited trait or a somatic mutation. Inone embodiment of the invention, the Pin1 marker is reduced Ser71phosphorylation of the Pin1 protein

The sample used for determining Pin1 expression can be selected from thegroup consisting of blood, urine, tissue biopsies, lymph, saliva,phlegm, cerebrospinal fluid, and pus. Furthermore, the sample can bederived from a diseased tissue, e.g., a tumor biopsy or fractionatedblood.

In one embodiment, the method of the invention can be used for treatingan immune disorder in a subject, e.g., a human subject, by administeringa Table 1 Compound to the subject in a therapeutically effective amount.The immune disorder can be any one or more selected from the groupconsisting of acne vulgaris; acute respiratory distress syndrome;Addison's disease; adrenocortical insufficiency; adrenogenital ayndrome;allergic conjunctivitis; allergic rhinitis; allergic intraocularinflammatory diseases, ANCA-associated small-vessel vasculitis;angioedema; ankylosing spondylitis; aphthous stomatitis; arthritis,asthma; atherosclerosis; atopic dermatitis; autoimmune disease;autoimmune hemolytic anemia; autoimmune hepatitis; Behcet's disease;Bell's palsy; berylliosis; bronchial asthma; bullous herpetiformisdermatitis; bullous pemphigoid; carditis; celiac disease; cerebralischaemia; chronic obstructive pulmonary disease; cirrhosis; Cogan'ssyndrome; contact dermatitis; COPD; Crohn's disease; Cushing's syndrome;dermatomyositis; diabetes mellitus; discoid lupus erythematosus;eosinophilic fasciitis; epicondylitis; erythema nodosum; exfoliativedermatitis; fibromyalgia; focal glomerulosclerosis; giant cellarteritis; gout; gouty arthritis; graft-versus-host disease; handeczema; Henoch-Schonlein purpura; herpes gestationis; hirsutism;hypersensitivity drug reactions; idiopathic cerato-scleritis; idiopathicpulmonary fibrosis; idiopathic thrombocytopenic purpura; inflammatorybowel or gastrointestinal disorders, inflammatory dermatoses; juvenilerheumatoid arthritis; laryngeal edema; lichen planus; Loeffler'ssyndrome; lupus nephritis; lupus vulgaris; lymphomatoustracheobronchitis; macular edema; multiple sclerosis; musculoskeletaland connective tissue disorder; myasthenia gravis; myositis; obstructivepulmonary disease; ocular inflammation; organ transplant rejection;osteoarthritis; pancreatitis; pemphigoid gestationis; pemphigusvulgaris; polyarteritis nodosa; polymyalgia rheumatica; primaryadrenocortical insufficiency; primary billiary cirrhosis; pruritusscroti; pruritis/inflammation, psoriasis; psoriatic arthritis; Reiter'sdisease; relapsing polychondritis; rheumatic carditis; rheumatic fever;rheumatoid arthritis; rosacea caused by sarcoidosis; rosacea caused byscleroderma; rosacea caused by Sweet's syndrome; rosacea caused bysystemic lupus erythematosus; rosacea caused by urticaria; rosaceacaused by zoster-associated pain; sarcoidosis; scleroderma; segmentalglomerulosclerosis; septic shock syndrome; serum sickness; shouldertendinitis or bursitis; Sjogren's syndrome; Still's disease;stroke-induced brain cell death; Sweet's disease; systemicdermatomyositis; systemic lupus erythematosus; systemic sclerosis;Takayasu's arteritis; temporal arteritis; thyroiditis; toxic epidermalnecrolysis; tuberculosis; type-1 diabetes; ulcerative colitis; uveitis;vasculitis; and Wegener's granulomatosis.

In another embodiment, the method of the invention can be used fortreating a proliferative disorder in a subject by administering a Table1 Compound to the subject in a therapeutically effective amount. Theproliferative disorder can be any one or more selected from the groupconsisting of acute leukemia, acute lymphocytic leukemia, acutemyelocytic leukemia, acute myeloblastic leukemia, acute promyelocyticleukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemi), Hodgkin's disease, non-Hodgkin's disease,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma,meningioma, melanoma, neuroblastoma, and retinoblastoma.

In one embodiment, the method of the invention may further include theadministration of a low dosage of a second therapeutic compound, e.g.,an anti-inflammatory compound, anti-microbial compound, anti-viralcompound, or an anti-cancer compound. The second therapeutic compoundcan be selected from the group consisting of corticosteroids, NSAIDs,COX-2 inhibitors, biologics, small molecule immunomodulators,non-steroidal immunophilin-dependent immunosuppressants, 5-aminosalicylic acid, DMARDs, hydroxychloroquine sulfate, and penicillamine.Alternatively, the second therapeutic compound can be selected from thegroup consisting of microtubule inhibitors, topoisomerase inhibitors,platins, alkylating agents, and anti-metabolites. The second therapeuticcompound can also be selected from the group consisting of1-D-ribofuranosyl-1,2,4-triazole-3 carboxamide, 9→2-hydroxy-ethoxymethylguanine, adamantanamine, 5-iodo-2′-deoxyuridine,trifluorothymidine, interferon, adenine arabinoside, proteaseinhibitors, thymidine kinase inhibitors, sugar or glycoprotein synthesisinhibitors, structural protein synthesis inhibitors, attachment andadsorption inhibitors, and nucleoside analogues such as acyclovir,penciclovir, valacyclovir, and ganciclovir.

In yet another embodiment, the second therapeutic compound can beselected from the group consisting of MK-2206, ON 013105, RTA 402, BI2536, Sorafenib, ISIS-STAT3Rx, a microtubule inhibitor, a topoisomeraseinhibitor, a platin, an alkylating agent, an anti-metabolite,paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide,5-fluorouracil, carboplatin, altretamine, aminoglutethimide, amsacrine,anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil,2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide,cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel,estramustine phosphate, floxuridine, fludarabine, gentuzumab,hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon,irinotecan, lomustine, mechlorethamine, melphalen, 6-mercaptopurine,methotrexate, mitomycin, mitotane, mitoxantrone, pentostatin,procarbazine, rituximab, streptozocin, tamoxifen, temozolomide,teniposide, 6-thioguanine, topotecan, trastuzumab, vincristine,vindesine, and/or vinorelbine.

In one embodiment, the invention features a pharmaceutical compositionof a Table 1 Compound. The pharmaceutical composition can be formulatedas a pill, ointment, cream, foam, capsule, or a liquid for administeringto a subject.

By “Table 1 Compound” is meant any of the compounds listed in Table 1,or any compound falling within the corresponding generic formula as setforth below.

TABLE 1 No. STRUCTURE Formula 1

(I) 2

(I) 3

(I) 4

(I) 5

(I) 6

(I) 7

(I) 8

(I) 9

(II) 10

(II) 11

12

13

(III) 14

(III) 15

(III) 16

17

(IV) 18

(IV) 19

20

21

22

23

24

25

26

27

28

(V) 29

(V) 30

(V) 31

(V) 32

(V) 33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

In some embodiments, the compound has a structure according to thefollowing formula,

or a stereoisomer thereof, or a pharmaceutically acceptable saltthereof, where

each of R₁, R₂, R₃, and R₄ is, independently, H, optionally substitutedC1-C6 alkyl, OH, optionally substituted C1-C6 alkoxy, halogen, nitro,optionally substituted C1-C6 acyl, or CO₂R₁₀;

each of R₅, R₆, and R₁₀ is, independently, H or optionally substitutedC1-C6 alkyl;

R_(7a) and R_(7b) are both H, or R_(7a) and R_(7b) combine to form acarbon-carbon double bond; and

each of R₈ and R₉ is, independently, H, optionally substituted C1-C6alkyl, OH, optionally substituted C1-C6 alkoxy, optionally substitutedaryloxy, SH, optionally substituted thioaryloxy, halogen, optionallysubstituted C1-C6 acyl.

In some embodiments, not more than one of R₁-R₄ can be nitro.

In some embodiments, at least one of R₁-R₄ is OH, halogen (e.g., F, Cl,or Br), optionally substituted C1-C6 alkyl (e.g., CH₃ or CF₃),optionally substituted C1-C6 acyl (e.g., CO₂Me) or CO₂R₁₀ (e.g., CO₂H).

In some embodiments, 1, 2, or 3 of R₁-R₄ is halogen (e.g., F, Cl, orBr).

In some embodiments, R₅ and R₆ are both H.

In some embodiments, R_(7a) and R_(7b) combine to form a carbon-carbondouble bond. In further embodiments, both R₈ and R₉ are H.

In some embodiments, R_(7a) and R_(7b) are both H.

In some embodiments, the compound is any of Compounds 1-8 of Table 1.

In some embodiments, the compound has a structure according to thefollowing formula,

or a stereoisomer thereof, or a pharmaceutically acceptable saltthereof, wherein

n is 0, 1, 2, 3, or 4;

each R₁, when present, is, independently, optionally substituted C1-C6alkyl, OH, optionally substituted C1-C6 alkoxy, halogen, nitro, oroptionally substituted C1-C6 acyl;

R₂ is H or optionally substituted C1-C6 alkyl; R_(3a) and R_(3b) areboth H, or R_(3a) and R_(3b) combine to form a carbon-oxygen doublebond;

R₄ and R₅ are both H, or R₄ and R₅ combine to form a carbon-carbondouble bond;

R₆ is optionally substituted phenyl; and

R₇ is optionally substituted C1-C6 alkyl.

In some embodiments, n is 0. In other embodiments, R_(3a) and R_(3b)combine to form a carbon-oxygen double bond. In still other embodiments,R₂ is H. In certain embodiments, R₄ and R₅ combine to form acarbon-carbon double bond. In other embodiments, R₇ is optionallysubstituted Cl alkyl (e.g., CH₂Cl). In other embodiments, R₆ is phenylhaving 1, 2, 3, 4, or 5 substituents (e.g., R₆ is tolyl).

In some embodiments, the compound is any of compounds 9-10 in table 1.

In some embodiments, the compound has a structure according to thefollowing formula,

or a stereoisomer thereof, or a pharmaceutically acceptable saltthereof, where

one of R₁ and R₂ is H, and the other is —NH(optionally substitutedphenyl); and

each of R₃, R₄, R₅, and R₆ is, independently, H, OR₇, or SO₃R₈;

each of R₇ and R₈ is, independently, H or optionally substituted C1-C6alkyl; and

wherein one and only one of R₃, R₄, R₅, and R₆ is SO₃R₈, and

wherein one and only one of R₃, R₄, R₅, and R₆ is OR₇.

In some embodiments, the optionally substituted phenyl has 1, 2, 3, 4,or 5 substituents. In other embodiments, the phenyl is unsubstituted.

In some embodiments, one of R₃ or R₆ is OH, and one of R₄ or R₅ is SO₃R₈(e.g., SO₃H).

In some embodiments, the compound is one of Compounds 13-15 of Table 1.

In some embodiments, the compound has a structure according to thefollowing formula,

or a stereoisomer thereof, or a pharmaceutically acceptable saltthereof, where

each of R₁ and R₂ is, independently, optionally substituted C1-C6 alkyl;and

A is a phenyl or 5-membered heteroaryl comprising a carboxyl substituentaccording to the substructure CO₂R₃, and where A comprises 0, 1, 2, or 3substituent groups.

In some embodiments, each of R₁ and R₂ is, independently, unsubstitutedC1-C6 alkyl (e.g., CH₃).

In other embodiments, the CO₂R₃ substituent is adjacent to the atom ofsubstructure A that is covalently attached to the pyrrole nitrogen. Inother embodiments, when A is phenyl, the CO₂R₃ substituent may be ortho,meta, or para to the pyrrole group.

In still other embodiments, R₃ is H.

In certain embodiments, A is phenyl or thienyl.

In some embodiments, the compound is any of compounds 17-19 of Table 1.

In still other embodiments, the compound has a structure according tothe following formula,

or a stereoisomer thereof, or a pharmaceutically acceptable saltthereof, where

R₁ is CN or C(═O)R₃; and

each R₂ and R₃ is, independently, optionally substituted phenyl or anoptionally substituted 5-to-6-membered heteroaryl.

In some embodiments, R₁ is C(═O)R₃. In further embodiments, both R₂ andR₃ are the same group. In some embodiments, both R₂ and R₃ are phenylhaving 0, 1, 2, or 3 substituents (e.g., methyl or methoxy). In otherembodiments, both R₂ and R₃ are optionally substituted five-memberedheteroaryls (e.g., optionally substituted pyrazolyl groups).

In some embodiments, R₁ is CN. In further embodiments, R₃ is anoptionally substituted five-membered heteroaryl group (e.g., thienyl).

In some embodiments, the compound is any of compounds 28-32 of Table 1.

As used herein, the term “C1-C6 alkoxy” represents a chemicalsubstituent of formula —OR, where R is an optionally substituted C1-C6alkyl group, unless otherwise specified. In some embodiments, the alkylgroup can be substituted, e.g., the alkoxy group can have 1, 2, 3, 4, 5or 6 substituent groups as defined herein.

As used herein, the term “C1-C6 acyl” refers to a C1-C6 alkyl group thatincludes a C(═O) moiety and which may be further substituted asdescribed herein.

As used herein, the term “alkyl,” “alkenyl” and “alkynyl” includestraight-chain, branched-chain and cyclic monovalent substituents, aswell as combinations of these, containing only C and H whenunsubstituted. Examples include methyl, ethyl, isobutyl, cyclohexyl,cyclopentylethyl, 2-propenyl, 3-butynyl, and the like. The term“cycloalkyl,” as used herein, represents a monovalent saturated orunsaturated non-aromatic cyclic alkyl group having between three to ninecarbons (e.g., a C3-C9 cycloalkyl), unless otherwise specified, and isexemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, bicyclo[2.2.1.]heptyl, and the like. When the cycloalkylgroup includes one carbon-carbon double bond, the cycloalkyl group canbe referred to as a “cycloalkenyl” group. Exemplary cycloalkenyl groupsinclude cyclopentenyl, cyclohexenyl, and the like.

Typically, the alkyl, alkenyl and alkynyl groups contain 1-12 carbons(e.g., C1-C12 alkyl) or 2-12 carbons (e.g., C2-C12 alkenyl or C2-C12alkynyl). In some embodiments, the alkyl groups are C1-C8, C1-C6, C1-C4,C1-C3, or C1-C2 alkyl groups; or C2-C8, C2-C6, C2-C4, or C2-C3 alkenylor alkynyl groups. Further, any hydrogen atom on one of these groups canbe replaced with a substituent as described herein.

“Aromatic” moiety or “aryl” moiety refers to any monocyclic or fusedring bicyclic system which has the characteristics of aromaticity interms of electron distribution throughout the ring system and includes amonocyclic or fused bicyclic moiety such as phenyl or naphthyl;“heteroaromatic” or “heteroaryl” also refers to such monocyclic or fusedbicyclic ring systems containing one or more heteroatoms selected fromO, S and N. The inclusion of a heteroatom permits inclusion of5-membered rings to be considered aromatic as well as 6-membered rings.Thus, typical aromatic/heteroaromatic systems include pyridyl,pyrimidyl, indolyl, benzimidazolyl, benzotriazolyl, isoquinolyl,quinolyl, benzothiazolyl, benzofuranyl, thienyl, furyl, pyrrolyl,thiazolyl, oxazolyl, isoxazolyl, benzoxazolyl, benzoisoxazolyl,imidazolyl and the like. Because tautomers are theoretically possible,phthalimido is also considered aromatic. Typically, the ring systemscontain 5-12 ring member atoms or 6-10 ring member atoms. In someembodiments, the aromatic or heteroaromatic moiety is a 6-memberedaromatic rings system optionally containing 1-2 nitrogen atoms. Moreparticularly, the moiety is an optionally substituted phenyl, pyridyl,indolyl, pyrimidyl, pyridazinyl, benzothiazolyl or benzimidazolyl,pyrazolyl, imidazolyl, isoxazolyl, thiazolyl, benzothiazolyl, indolyl.Even more particularly, such moiety is phenyl, pyridyl, or pyrimidyl andeven more particularly, it is phenyl.

As used herein, the term “aryloxy” refers to aromatic or heteroaromaticsystems which are coupled to another residue through an oxygen atom. Atypical example of an O-aryl is phenoxy. Similarly, “thioaryloxy” refersto aromatic or heteroaromatic systems which are coupled to anotherresidue through a sulfur atom.

As used herein, a halogen is selected from F, Cl, Br, and I, and moreparticularly it is fluoro or chloro.

In general, a substituent group (e.g., alkyl, alkenyl, alkynyl, or aryl(including all heteroforms defined above) may itself optionally besubstituted by additional substituents. The nature of these substituentsis similar to those recited with regard to the substituents on the basicstructures above. Thus, where an embodiment of a substituent is alkyl,this alkyl may optionally be substituted by the remaining substituentslisted as substituents where this makes chemical sense, and where thisdoes not undermine the size limit of alkyl per se; e.g., alkylsubstituted by alkyl or by alkenyl would simply extend the upper limitof carbon atoms for these embodiments, and is not included. For example,where a group is substituted, the group may be substituted with 1, 2, 3,4, 5, or 6 substituents. Optional substituents include, but are notlimited to: C1-C6 alkyl or heteroaryl, C2-C6 alkenyl or heteroalkenyl,C2-C6 alkynyl or heteroalkynyl, halogen; aryl, heteroaryl, azido(—N₃),nitro (—NO₂), cyano (—CN), acyloxy(—OC(═O)R′), acyl (—C(═O)R′), alkoxy(—OR′), amido (—NR′C(═O)R″ or —C(═O)NRR′), amino (—NRR′), carboxylicacid (—CO₂H), carboxylic ester (—CO₂R′), carbamoyl (—OC(═O)NR′R″ or—NRC(═O)OR′), hydroxy (—OH), isocyano (—NC), sulfonate (—S(═O)₂OR),sulfonamide (—S(═O)₂NRR′ or —NRS(═O)₂R′), or sulfonyl (—S(═O)₂R), whereeach R or R′ is selected, independently, from H, C1-C6 alkyl orheteroaryl, C2-C6 alkenyl or heteroalkenyl, 2C-6C alkynyl orheteroalkynyl, aryl, or heteroaryl. A substituted group may have, forexample, 1, 2, 3, 4, 5, 6, 7, 8, or 9 substituents.

Typical optional substituents on aromatic or heteroaromatic groupsinclude independently halo, CN, NO₂, CF₃, OCF₃, COOR′, CONR′₂, OR′, SR′,SOR′, SO₂R′, NR′₂, NR′(CO)R′,NR′C(O)OR′, NR′C(O)NR′₂, NR′SO₂NR′₂, orNR′SO₂R′, wherein each R′ is independently H or an optionallysubstituted group selected from alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, heteroaryl, and aryl (all as definedabove); or the substituent may be an optionally substituted groupselected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,heteroalkynyl, aryl, heteroaryl, O-aryl, O-heteroaryl and arylalkyl.

Optional substituents on a non-aromatic group (e.g., alkyl, alkenyl, andalkynyl groups), are typically selected from the same list ofsubstituents suitable for aromatic or heteroaromatic groups, except asnoted otherwise herein. A non-aromatic group may also include asubstituent selected from ═O and ═NOR′ where R′ is H or an optionallysubstituted group selected from alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteralkynyl, heteroaryl, and aryl (all as definedabove).

The term “pharmaceutically acceptable salt,” as use herein, representsthose salts of the compounds described here (e.g., a compound accordingto any of Formulas (I)-(V) or any of Compounds (1)-(50) of Table 1) thatare, within the scope of sound medical judgment, suitable for use incontact with the tissues of humans and animals without undue toxicity,irritation, allergic response and the like and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. For example, pharmaceutically acceptable saltsare described in: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P. H.Stahl and C. G. Wermuth), Wiley-VCH, 2008. The salts can be prepared insitu during the final isolation and purification of the compoundsdescribed herein or separately by reacting the free base group with asuitable organic acid.

The compounds of the invention may have ionizable groups so as to becapable of preparation as pharmaceutically acceptable salts. These saltsmay be acid addition salts involving inorganic or organic acids or thesalts may, in the case of acidic forms of the compounds of the inventionbe prepared from inorganic or organic bases. Frequently, the compoundsare prepared or used as pharmaceutically acceptable salts prepared asaddition products of pharmaceutically acceptable acids or bases.Suitable pharmaceutically acceptable acids and bases are well-known inthe art, such as hydrochloric, sulphuric, hydrobromic, acetic, lactic,citric, or tartaric acids for forming acid addition salts, and potassiumhydroxide, sodium hydroxide, ammonium hydroxide, caffeine, variousamines, and the like for forming basic salts. Methods for preparation ofthe appropriate salts are well-established in the art.

Representative acid addition salts include acetate, adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts andthe like. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamineand the like.

In some cases, the compounds of the invention contain one or more chiralcenters. The invention includes each of the isolated stereoisomericforms as well as mixtures of stereoisomers in varying degrees of chiralpurity, including racemic mixtures. It also encompasses the variousdiastereomers and tautomers that can be formed.

The compounds described herein can be prepared according to conventionalmeans known in the art.

By the term “immune disorder” is meant a disorder characterized byderegulation of Toll like receptor and/or type 1 interferon.

By the term “proliferative disorder” is meant a disorder characterizedby inappropriate accumulation of a cell population in a tissue (e.g., byabnormal cell growth). This inappropriate accumulation may be the resultof a genetic or epigenetic variation that occurs in one or more cells ofthe cell population. This genetic or epigenetic variation causes thecells of the cell population to grow faster, die slower, ordifferentiate slower than the surrounding, normal tissue. The cellpopulation includes cells of hematopoietic, epithelial, endothelial, orsolid tissue origin.

As used herein, the term “Pin1 marker” refers to a marker which iscapable of being indicative of Pin1 activity levels in an organism or asample of the invention. Pin1 markers include nucleic acid molecules(e.g., mRNA, DNA) which correspond to some or all of a Pin1 gene,peptide sequences (e.g., amino acid sequences) which correspond to someor all of a Pin1 protein, nucleic acid sequences which are homologous toPin1 gene sequences, peptide sequences which are homologous to Pin1peptide sequences, antibodies to Pin1 protein, substrates of Pin1protein, binding partners of Pin1 protein, and activity of Pin1.

By “elevated levels of a Pin1 marker” is meant a level of Pin1 markerthat is altered thereby indicating elevated Pin1 activity. “Elevatedlevels of a Pin1 marker” include levels at least 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%,1000%, or greater than, or 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100% less than the marker levels measuredin a normal, disease fee subject or tissue.

By “corticosteroid” is meant any naturally occurring or syntheticsteroid hormone which can be derived from cholesterol and ischaracterized by a hydrogenated cyclopentanoperhydrophenanthrene ringsystem. Naturally occurring corticosteroids are generally produced bythe adrenal cortex. Synthetic corticosteroids may be halogenated.Functional groups required for activity include a double bond at A4, aC3 ketone, and a C20 ketone. Corticosteroids may have glucocorticoidand/or mineralocorticoid activity. In preferred embodiments, thecorticosteroid is either fludrocortisone or prednisolone.

Exemplary corticosteroids include algestone, 6-alpha-fluoroprednisolone,6-alpha-methylprednisolone, 6-alpha-methylprednisolone 21-acetate,6-alpha-methylprednisolone 21-hemisuccinate sodium salt,6-alpha,9-alpha-difluoroprednisolone 21-acetate 17-butyrate, amcinafal,beclomethasone, beclomethasone dipropionate, beclomethasone dipropionatemonohydrate, 6-beta-hydroxycortisol, betamethasone,betamethasone-17-valerate, budesonide, clobetasol, clobetasolpropionate, clobetasone, clocortolone, clocortolone pivalate, cortisone,cortisone acetate, cortodoxone, deflazacort, 21-deoxycortisol,deprodone, descinolone, desonide, desoximethasone, dexamethasone,dexamethasone-21-acetate, dichlorisone, diflorasone, diflorasonediacetate, diflucortolone, doxibetasol, fludrocortisone, flumethasone,flumethasone pivalate, flumoxonide, flunisolide, fluocinonide,fluocinolone acetonide, 9-fluorocortisone, fluorohydroxyandrostenedione,fluorometholone, fluorometholone acetate, fluoxymesterone, flupredidene,fluprednisolone, flurandrenolide, formocortal, halcinonide,halometasone, halopredone, hyrcanoside, hydrocortisone, hydrocortisoneacetate, hydrocortisone butyrate, hydrocortisone cypionate,hydrocortisone sodium phosphate, hydrocortisone sodium succinate,hydrocortisone probutate, hydrocortisone valerate,6-hydroxydexamethasone, isoflupredone, isoflupredone acetate,isoprednidene, meclorisone, methylprednisolone, methylprednisoloneacetate, methylprednisolone sodium succinate, paramethasone,paramethasone acetate, prednisolone, prednisolone acetate, prednisolonemetasulphobenzoate, prednisolone sodium phosphate, prednisolonetebutate, prednisolone-21-hemisuccinate free acid,prednisolone-21-acetate, prednisolone-21(beta-D-glucuronide),prednisone, prednylidene, procinonide, tralonide, triamcinolone,triamcinolone acetonide, triamcinolone acetonide 21-palmitate,triamcinolone diacetate, triamcinolone hexacetonide, and wortmannin.Desirably, the corticosteroid is fludrocortisone or prednisolone.

“Treatment,” as used herein, refers to the application or administrationof a therapeutic agent (e.g., a Table 1 Compound) to a patient, orapplication or administration of a therapeutic agent to an isolatedtissue or cell line from a patient, who has a disease, a symptom ofdisease or a predisposition toward a disease, with the purpose to cure,heal, alleviate, relieve, alter, remedy, ameliorate, improve or affectthe disease, the symptoms of disease or the predisposition towarddisease, or to slow the progression of the disease.

As used herein, the terms “sample” and “biological sample” includesamples obtained from a mammal or a subject containing Pin1 which can beused within the methods described herein, e.g., tissues, cells andbiological fluids isolated from a subject, as well as tissues, cells andfluids present within a subject. Typical samples from a subject includetissue samples, tumor samples, blood, urine, biopsies, lymph, saliva,phlegm, cerebrospinal fluid, pus, and the like. The sample can be from adiseased tissue such as a tumor biopsy or fractionated blood.

By a “low dosage” or “low concentration” is meant at least 5% less(e.g., at least 10%, 20%, 50%, 80%, 90%, or even 95%) than the loweststandard recommended dosage or lowest standard recommended concentrationof a particular compound formulated for a given route of administrationfor treatment of any human disease or condition. For example, a lowdosage of an anti-inflammatory, anti-microbial, or anti-viral compoundformulated for oral administration will differ from a low dosage of ananti-inflammatory, anti-microbial, or anti-viral compound formulated forintravenous administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a Pin1 competition fluorescencepolarization assay. Pin1 protein is incubated in the presence of afluorophore-labeled Pin1 probe with which Pin1 may interact and,optionally, a compound of interest. The compound may inhibit theinteraction of Pin1 with the fluorophore-labeled probe. When thefluorophore on the probe is excited by polarized light, the resultingemission of polarized light will depend upon the extent to which theprobe interacts with Pin1. A compound that does not inhibit theinteraction of the probe with Pin1 generally will not decrease thedetected emission of polarized light relative to a control value such asthe emission of polarized fluorescence by an uninhibited control. Incontrast, when the compound does inhibit interaction of probe and Pin1,the detected emission of polarized light will be decreased relative to acontrol value such as the emission of polarized fluorescence by anuninhibited control.

FIG. 2 is a set of three graphs relating to the performance of a Pin1competition fluorescence polarization assay of 1607 plates, eachcontaining 1536 wells, covering a total of 393,181 compounds. Graphsshow S:B ratio, Z′ Score (Z′ factor=1−3*(SD of positive control+SD ofbasal)/(median of positive control−median of basal)), and CV % (CV(coefficient of variation)=SD of compound area/median of compound area).

Each of FIGS. 3A-3D is a series of compounds tested in a Pin1competition fluorescence polarization assay together with the chemicalstructures of a subset of these compounds and curves relating to theperformance of those compounds for which chemical structures are shownin the assay. A set of assayed compounds is identified in the list shownon the left of each of FIGS. 3A-3D. Chemical structures of a subset ofthe listed compounds are shown to the right of the list, and theperformance each compound for which a chemical structure is shown isvisualized in the two curves to the right of the chemical structure. Thecurve closest to each chemical structure shows the relationship betweenlog concentration (M) of the compound (X axis) and the emission ofpolarized fluorescence (Y axis), shown as percentage change incomparison to the emission of polarized fluorescence by an uninhibitedcontrol. The rightmost set of curves shows the relationship between logconcentration (M) of the compound (X axis) and total fluorescenceintensity (Y axis). Pin1 inhibitors in FIG. 3A demonstrate nointerference of total fluorescence. Pin1 inhibitors in FIG. 3B maydemonstrate interference of total fluorescence. Pin1 inhibitors in FIG.3C demonstrate strong interference of total fluorescence. Pin1inhibitors in FIG. 3D possibly quench total fluorescence.

FIG. 4 is a series of curves that represent data from a Pin1 competitionfluorescence polarization assay and that have been grouped intocategories based on curve characteristics. Each curve plots logconcentration (M) of the compound (X axis) against the emission ofpolarized fluorescence (Y axis), shown as percentage change incomparison to the emission of polarized fluorescence by an uninhibitedcontrol. In three dimensional plots, the third dimension is the samplenumber. Class 1 includes complete curves, having 2 asymptotes andr²≧0.9, with an efficacy of either >80% (class 1.1) or ≦80% (class 1.2).Class 1 curves may also be classified as noisy curves when efficacyis >80% and r²<0.9 (class 1.3) or when efficacy is ≦80% and r²<0.9(class 1.4). Class 2 includes incomplete curves having 1 asymptote andan r² value of either >0.9 (subclass a) or <0.9 (subclass b), with anefficacy of either >80% (class 2.1) or ≦80% (class 2.2). Class 2 curvesmay also be classified as noisy curves when efficacy is >80% and r²<0.9(class 2.3) or when efficacy is ≦80% and r²<0.9 (class 2.4). Class 3includes single point activity curves having 1 asymptote and anefficacy >3 SD from the mean activity of the sample field at the highesttested concentration. Class 4 includes inactive curves, for which thereare no asymptotes and for which efficacy and r² values are notapplicable. A fifth class captures any curves not otherwise classified.

FIG. 5 summarizes the occurrence of curve types identified in a Pin1competition fluorescence polarization assay. Active inhibitors are thoseyielding curves that fall into classes 1 and 2 with efficacy >50%. Class4 curves are classified as inactive. All other curves are classified asinconclusive. Of 2315 active inhibitors, those demonstrating stronginterference of fluorescence were not selected for further assessment.

DETAILED DESCRIPTION OF THE INVENTION

We have discovered compounds (i.e., the compounds of Table 1) useful forinhibiting Pin1. Inhibitors of Pin1 are useful for treating immunedisorders and proliferative disorders (e.g., disorders characterized byelevated Pin1 marker levels). In other preferred embodiments, thecompounds of Table 1 are useful for administration to subjects havingaging-related disorders, asthma, and microbial infections. Theusefulness of Pin1 inhibitors in treating the above diseases is furtheridentified or elaborated in PCT Application Nos. PCT/US2012/029077,PCT/US2012/35473, PCT/US2012/39850, PCT/US2010/054077, U.S. ApplicationPublication No. 2008/0214470 A1, U.S. Pat. Nos. 6,495,376 B1, 6,462,173B1, 8,129,131 B2, 8,258,099 B2, and U.S. Provisional Application No.61/490,338, each of which is hereby specifically incorporated byreference in its entirety.

The compounds of Table 1 were identified and validated using the methodsdescribed in the examples. In brief, the compounds were identified in ahigh-throughput screen for displacing a known binder of Pin-1 from Pin-1proteins. Based on this displacement, and the nature of the displacedmolecule, we conclude that the compounds are likely to inhibit Pin-1activity.

I. PIN1

Phosphorylation on serine/threonine-proline motifs restrains cis/transprolyl isomerization, and also creates a binding site for the essentialprotein Pin1. Pin1 binds and regulates the activity of a defined subsetof phosphoproteins, as well as participating in the timing of mitoticprogression. Both structural and functional analyses have indicated thatPin1 contains a phosphoserine/threonine-binding module that bindsphosphoproteins, and a catalytic activity that specifically isomerizesthe phosphorylated phosphoserinelthreonine-proline. Both of these Pin1activities are essential for Pin1 to carry out its function in vivo.

Pin1 has previously been shown to act on IRF3 to affect IFN-β productionupon TLR3 or RIG-I activation. However, recent results have shown thatunlike IRF3- or TLR3-deficient mice, IRF7 or IRAK1-deficient micecompletely fail to mount a type I IFN antiviral responses due to loss oftype I IFN secretion from pDCs. Results have uncovered an essential rolefor Pin1 as a novel regulator of IRAK1 activation in TLR signaling andtype I IFN-mediated innate and adaptive immunity and revealed that Pin1inhibitors, which are under active development, may represent a noveltherapeutic approach that would allow selective inhibition of the type IIFN response while leaving proinflammatory cytokine productionunaffected.

Pin1 is highly conserved and contains a protein-interacting module,called WW domain, and a catalytically active peptidyl-prolyl isomerase(PPIase). Pin1 is structurally and functionally distinct from members oftwo other well-characterized families of PPIases, the cyclophilins andthe FKBPs. PPIases are ubiquitous enzymes that catalyze the typicallyslow prolyl isomerization of proteins, allowing relaxation of localenergetically unfavorable conformational states. Phosphorylation onSer/Thr residues immediately preceding Pro not only alters the prolylisomerization rate, but also creates a binding site for the WW domain ofPin1. The WW domain acts a novel phosphoserine-binding module targetingPin1 to a highly conserved subset of phosphoproteins. Furthermore, Pin1displays a unique phosphorylation-dependent PPIase that specificallyisomerizes phosphorylated Ser/Thr-Pro bonds and regulates the functionof phosphoproteins.

II. METHODS OF IDENTIFYING PIN1 INHIBITORS

Numerous methods of identifying a Pin1 inhibitor are known in the art.In one method of identifying a Pin1 inhibitor, candidate or testcompounds are substrates of a Pin1 protein or polypeptide orbiologically active portion thereof that can bind to a Pin1 protein orpolypeptide or biologically active portion thereof. In another method ofidentifying a Pin1 inhibitor,

Test compounds that may be screened to identify a Pin1 inhibitor can beobtained from numerous available resources or using any of the numerousapproaches in combinatorial library methods known in the art, including:biological libraries; spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam (1997) Anticancer Drug Des.12:145). Examples of methods for the synthesis of molecular librariescan be found in the art, for example in: DeWitt et al. (1993) Proc.Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad.Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Choet al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int.Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382;Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

In another method of identifying a Pin1 inhibitor, the assay is acell-based assay in which a cell expressing a Pin1 target molecule(e.g., a Pin1 substrate; a phosphoprotein) is contacted with a testcompound and the ability of the test compound to inhibit the activity ofthe Pin1 target molecule is determined. Determining the ability of thetest compound to modulate the activity of a Pin1 target molecule can beaccomplished, for example, by determining the ability of the Pin1protein to bind to or interact with the Pin1 target molecule, or bydetermining the ability of the Pin1 protein to isomerize the Pin1 targetmolecule.

Determining the ability of the Pin1 protein to bind to or interact witha Pin1 target molecule can be accomplished by determining directbinding. Determining the ability of the Pin1 protein to bind to orinteract with a Pin1 target molecule can be accomplished, for example,by coupling the Pin1 protein with a radioisotope or enzymatic label suchthat binding of the Pin1 protein to a Pin1 target molecule can bedetermined by detecting the labeled Pin1 protein in a complex. Forexample, Pin1 molecules, e.g., Pin1 proteins, can be labeled with, I¹²⁵,S³⁵, C¹⁴, or H³, either directly or indirectly, and the radioisotopedetected by direct counting of radioemmission or by scintillationcounting. Alternatively, Pin1 molecules can be enzymatically labeledwith, for example, horseradish peroxidase, alkaline phosphatase, orluciferase, and the enzymatic label detected by determination ofconversion of an appropriate substrate to product.

The ability of a compound to modulate the interaction between Pin1 andits target molecule may also be determined without the labeling any ofthe interactants. For example, a microphysiometer can be used to detectthe interaction of Pin1 with its target molecule without the labeling ofeither Pin1 or the target molecule (McConnell (1992) Science257:1906-1912). A “microphysiometer” (e.g., Cytosensor) is an analyticalinstrument that measures the rate at which a cell acidifies itsenvironment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between compound and receptor.

Determining the ability of the Pin1 protein to bind to or interact witha Pin1 target molecule can be accomplished by determining the activityof the target molecule. For example, the activity of the target moleculecan be determined by detecting induction of a downstream event (e.g.,expression of cyclin D1, mitosis etc.), detecting catalytic/enzymaticactivity of the target on an appropriate substrate, detecting theinduction of a reporter gene (comprising a target-responsive regulatoryelement (e.g., AP-1) operatively linked to a nucleic acid optionallyencoding a detectable marker, e.g., chloramphenicol acetyl transferase),or detecting a target-regulated cellular response.

Another method identifying a Pin1 inhibitor utilizes a cell-free assayin which a Pin1 protein or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tobind to the Pin1 protein or biologically active portion thereof isdetermined. Binding of the test compound to the Pin1 protein can bedetermined either directly or indirectly. For instance, the assay mayinclude contacting the Pin1 protein or biologically active portionthereof with a known compound which binds Pin1 to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with a Pin1 protein, whereindetermining the ability of the test compound to interact with a Pin1protein comprises determining the ability of the test compound topreferentially bind to Pin1 or a biologically active portion thereof ascompared to the known compound.

In another method of identifying a Pin1 inhibitor, the assay is acell-free assay in which a Pin1 protein or biologically active portionthereof is contacted with a test compound and the ability of the testcompound to inhibit the activity of the Pin1 protein or biologicallyactive portion thereof is determined. Determining the ability of thetest compound to modulate the activity of a Pin1 protein can beaccomplished, for example, by determining the ability of the Pin1protein to bind to a Pin1 target molecule using a technology such asreal-time Biomolecular Interaction Analysis (BIA). Sjolander, S. andUrbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995)Curr. Opin. Struct. Biol. 5:699-705. As used herein, “BIA” is atechnology for studying biospecific interactions in real time, withoutlabeling any of the interactants (e.g., BIAcore). Changes in the opticalphenomenon of surface plasmon resonance (SPR) can be used as anindication of real-time reactions between biological molecules.

In another method of identifying a Pin1 inhibitor, determining theability of the test compound to modulate the activity of a Pin1 proteincan be accomplished by determining the ability of the Pin1 protein tofurther modulate the isomerization of the activity of a Pin1 targetmolecule (e.g., a Pin1 substrate, a phosphoprotein).

In another method of identifying a Pin1 inhibitor, the cell-free assayinvolves contacting a Pin1 protein or biologically active portionthereof with a known compound which binds the Pin1 protein to form anassay mixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to interact with the Pin1protein, wherein determining the ability of the test compound tointeract with the Pin1 protein comprises determining the ability of thePin1 protein to preferentially bind to or modulate the activity of aPin1 target molecule.

Cell-free assays for identifying a Pin1 inhibitor are amenable to use ofboth soluble and/or membrane-bound forms of proteins (e.g., Pin1proteins or biologically active portions thereof, or receptors to whichPin1 binds). In the case of cell-free assays in which a membrane-boundform of a protein is used (e.g., a cell surface Pin1 receptor) it may bedesirable to utilize a solubilizing agent such that the membrane-boundform of the protein is maintained in solution. Examples of suchsolubilizing agents include non-ionic detergents such asn-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether).sub.n,3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In some methods of identifying a Pin1 inhibitor, it may be desirable toimmobilize either Pin1 or its target molecule to facilitate separationof complexed from uncomplexed forms of one or both of the proteins, aswell as to accommodate automation of the assay. Binding of a testcompound to a Pin1 protein, or interaction of a Pin1 protein with atarget molecule in the presence and absence of a candidate compound, canbe accomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtitre plates, test tubes, andmicro-centrifuge tubes.

A fusion protein can be provided which adds a domain that allows one orboth of the proteins to be bound to a matrix. For example,glutathione-S-transferase/Pin1 fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or Pin1 protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, orthat are matrix immobilized in the case of beads, and complex formationmay be determined either directly or indirectly, for example, asdescribed above. Alternatively, the complexes can be dissociated fromthe matrix, and the level of Pin1 binding or activity determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin an assay for identifying a Pin1 inhibitor. For example, either a Pin1protein or a Pin1 target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated Pin1 protein ortarget molecules can be prepared from biotin-NHS(N-hydroxy-succinimide)using techniques well known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies reactive with Pin1 protein or target molecules but which donot interfere with binding of the Pin1 protein to its target moleculecan be derivatized to the wells of the plate, and unbound target or Pin1protein trapped in the wells by antibody conjugation. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the Pin1 protein or target molecule, as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the Pin1 protein or target molecule.

In other assays for identifying a Pin1 inhibitor, a cell is contactedwith a candidate compound and the expression of Pin1 mRNA or protein inthe cell is determined. The level of expression of Pin1 mRNA or proteinin the presence of the candidate compound is compared to the level ofexpression of Pin1 mRNA or protein in the absence of the candidatecompound. The candidate compound can then be identified as a modulatorof Pin1 expression based on this comparison. For example, whenexpression of Pin1 mRNA or protein is less (statistically significantlyless) in the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of Pin1 mRNA or proteinexpression.

In some assays for identifying a Pin1 inhibitor, Pin1 proteins can beused as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with Pin1 (“Pin1-binding proteins” or “Pin1-bp”).

A two-hybrid system may be used in an assay for identifying a Pin1inhibitor. A two-hybrid system may be based on the modular nature ofmost transcription factors and utilizes two different DNA constructs. Inone construct, the gene that codes for a Pin1 protein is fused to a geneencoding the DNA binding domain of a known transcription factor (e.g.,GAL-4). In the other construct, a DNA sequence, from a library of DNAsequences, that encodes an unidentified protein (“prey” or “sample”) isfused to a gene that codes for the activation domain of the knowntranscription factor. If the “bait” and the “prey” proteins are able tointeract, in vivo, forming a Pin1-dependent complex, the DNA-binding andactivation domains of the transcription factor are brought into closeproximity. This proximity allows transcription of a reporter gene (e.g.,LacZ) which is operably linked to a transcriptional regulatory siteresponsive to the transcription factor. Expression of the reporter genecan be detected and cell colonies containing the functionaltranscription factor can be isolated and used to obtain the cloned genewhich encodes the protein which interacts with the Pin1 protein.

III. MEASUREMENT OF PIN1 MARKER LEVELS

The present invention pertains to the treatment of disorders identifiedas coinciding with elevated Pin1 marker levels with Table 1 Compounds.In some aspects, the invention features the determination of Pin1 markerlevels in a subject and subsequently administering Table 1 Compound insubjects where Pin1 marker levels are determined to be elevated. Inother aspects, the invention can also feature the measurement of Pin1marker levels subsequent to the administration of Table 1 Compounds inorder to evaluate the progress of therapy in treating the immunedisorder or proliferative disorder.

Accordingly, one aspect of the present invention relates to diagnosticassays for measuring levels of Pin1 marker, as well as Pin1 activity, inthe context of a biological sample (e.g., blood, urine, biopsies, lymph,saliva, phlegm, and pus) to thereby determine whether an individual is acandidate for treatment with a Table 1 Compound. The invention featuresboth treatment of subjects exhibiting symptoms of an immune disorder, orproliferative disorder, and individuals at risk for developing such adisorder.

Diagnostic Assays

An exemplary method for detecting the presence or absence of Pin1protein or nucleic acid in a biological sample involves obtaining abiological sample from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting Pin1 protein ora nucleic acid (e.g., mRNA, genomic DNA) that encodes Pin1 protein suchthat the presence of Pin1 protein or nucleic acid is detected in thebiological sample. A preferred agent for detecting Pin1 mRNA or genomicDNA is a labeled nucleic acid probe capable of hybridizing to Pin1 mRNAor DNA. The nucleic acid probe can be, for example, a Pin1 nucleic acidor a corresponding nucleic acid such as an oligonucleotide of at least15, 30, 50, 100, 250 or 500 nucleotides in length which is capable ofspecifically hybridizing under stringent conditions to Pin1 mRNA orgenomic DNA. Other suitable probes for use in the diagnostic assays ofthe invention are described herein.

A preferred agent for detecting Pin1 marker is an antibody capable ofbinding to Pin1 protein, preferably an antibody with a detectable label.Antibodies can be polyclonal, or more preferably, monoclonal. An intactantibody, or a fragment thereof (e.g., Fab or F(ab′2) can be used. Theterm “labeled,” with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin.

With respect to antibody-based detection techniques, one of skill in theart can raise anti-Pin1 antibodies against an appropriate immunogen,such as isolated and/or recombinant Pin1 or a portion or fragmentthereof (including synthetic molecules, such as synthetic peptides)using no more than routine experimentation. Synthetic peptides can bedesigned and used to immunize animals, such as rabbits and mice, forantibody production. The nucleic and amino acid sequence of Pin1 isknown (Hunter et al., WO 97/17986 (1997); Hunter et al., U.S. Pat. Nos.5,952,467 and 5,972,697, the teachings of all of which are herebyincorporated by reference in their entirety) and can be used to designnucleic acid constructs for producing proteins for immunization or innucleic acid detection methods or for the synthesis of peptides forimmunization.

Conditions for incubating an antibody with a test sample can varydepending upon the tissue or cellular type. Incubation conditions candepend on the format employed in the assay, the detection methodsemployed, and the type and nature of the antibody used in the assay. Oneskilled in the art will recognize that any one of the commonly availableimmunological assay formats (such as radioimmunoassays, enzyme-linkedimmunosorbent assays, diffusion based Ouchterlony, or rocketimmunofluorescent assays) can readily be adapted to employ a Pin1antibody. Examples of such assays can be found in Chard, “AnIntroduction to Radioimmunoas say and Related Techniques,” ElsevierScience Publishers, Amsterdam, The Netherlands (1986); Bullock et al.,“Techniques in Immunocytochemistry,” Academic Press, Orlando, Fla. Vol.1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, “Practice and Theory ofenzyme Immunoassays: Laboratory Techniques in Biochemistry and MolecularBiology,” is Elsevier Science Publishers, Amsterdam, The Netherlands(1985).

The detection method of the invention can be used to detect Pin1 mRNA,protein, or genomic DNA in a biological sample in vitro as well as invivo. For example, in vitro techniques for detection of Pin1 mRNAinclude northern blot hybridizations and in situ hybridizations. Invitro techniques for detection of Pin1 protein include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations,immunofluorescence, or quantitative sequencing reactions. In vitrotechniques for detection of Pin1 genomic DNA include Southernhybridizations. The detection of genomic mutations in Pin1 (or othergenes that effect Pin1 marker levels) can be used to identify inheritedor somatic mutations. Furthermore, in vivo techniques for detection ofPin1 protein include introducing into a subject a labeled anti-Pin1antibody. For example, the antibody can be labeled with a radioactivemarker whose presence and location in a subject can be detected bystandard imaging techniques.

In another embodiment, the biological sample contains protein moleculesfrom the test subject. Alternatively, the biological sample can containmRNA molecules from the test subject or genomic DNA molecules from thetest subject. A preferred biological sample is a serum sample isolatedby conventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting Pin1 marker such that thepresence of Pin1 marker is detected in the biological sample, andcomparing the presence of Pin1 marker in the control sample with thepresence of Pin1 marker in the test sample.

The immunological assay test samples of the present invention mayinclude cells, protein or membrane extracts of cells, blood orbiological fluids such as ascites fluid or cerebrospinal fluid. The testsample used in the above-described method is based on the assay format,nature of the detection method and the tissues, cells or extracts usedas the sample to be assayed. Methods for preparing protein extracts ormembrane extracts of cells are well known in the art and can be readilybe adapted in order to obtain a sample which is compatible with thesystem utilized.

The invention also encompasses kits for detecting the presence of Pin1in a biological sample. For example, the kit can comprise a labeledcompound or agent capable of detecting Pin1 protein or mRNA in abiological sample; means for determining the amount of Pin1 in thesample; and means for comparing the amount of Pin1 in the sample with aknown standard. The compound or agent can be packaged in a suitablecontainer. The kit can further comprise instructions for using the kitto detect Pin1 protein or nucleic acid.

Pin1 marker levels can also be measured in an assay designed to evaluatea panel of target genes, e.g., a microarray or multiplex sequencingreaction. In the embodiments of the invention described herein, wellknown biochemical methods such as northern blot analysis, RNaseprotection assays, southern blot analysis, western blot analysis, insitu hybridization, immunocytochemical procedures of tissue sections orcellular spreads, and nucleic acid amplification reactions (e.g.,polymerase chain reactions) may be used interchangeably. One of skill inthe art would be capable of performing these well-established protocolsfor the methods of the invention. (See, for example, Ausubel, et al.,“Current Protocols in Molecular Biology,” John Wiley & Sons, NY, N.Y.(1999)).

Diagnostic assays can be carried out in, e.g., subjects diagnosed or atrisk of an immune disorder. Such disorders include, without limitation,acne vulgaris; acute respiratory distress syndrome; Addison's disease;adrenocortical insufficiency; adrenogenital ayndrome; allergicconjunctivitis; allergic rhinitis; allergic intraocular inflammatorydiseases, ANCA-associated small-vessel vasculitis; angioedema;ankylosing spondylitis; aphthous stomatitis; arthritis, asthma;atherosclerosis; atopic dermatitis; autoimmune disease; autoimmunehemolytic anemia; autoimmune hepatitis; Behcet's disease; Bell's palsy;berylliosis; bronchial asthma; bullous herpetiformis dermatitis; bullouspemphigoid; carditis; celiac disease; cerebral ischaemia; chronicobstructive pulmonary disease; cirrhosis; Cogan's syndrome; contactdermatitis; COPD; Crohn's disease; Cushing's syndrome; dermatomyositis;diabetes mellitus; discoid lupus erythematosus; eosinophilic fasciitis;epicondylitis; erythema nodosum; exfoliative dermatitis; fibromyalgia;focal glomerulosclerosis; giant cell arteritis; gout; gouty arthritis;graft-versus-host disease; hand eczema; Henoch-Schonlein purpura; herpesgestationis; hirsutism; hypersensitivity drug reactions; idiopathiccerato-scleritis; idiopathic pulmonary fibrosis; idiopathicthrombocytopenic purpura; inflammatory bowel or gastrointestinaldisorders, inflammatory dermatoses; juvenile rheumatoid arthritis;laryngeal edema; lichen planus; Loeffler's syndrome; lupus nephritis;lupus vulgaris; lymphomatous tracheobronchitis; macular edema; multiplesclerosis; musculoskeletal and connective tissue disorder; myastheniagravis; myositis; obstructive pulmonary disease; ocular inflammation;organ transplant rejection; osteoarthritis; pancreatitis; pemphigoidgestationis; pemphigus vulgaris; polyarteritis nodosa; polymyalgiarheumatica; primary adrenocortical insufficiency; primary billiarycirrhosis; pruritus scroti; pruritis/inflammation, psoriasis; psoriaticarthritis; Reiter's disease; relapsing polychondritis; rheumaticcarditis; rheumatic fever; rheumatoid arthritis; rosacea caused bysarcoidosis; rosacea caused by scleroderma; rosacea caused by Sweet'ssyndrome; rosacea caused by systemic lupus erythematosus; rosacea causedby urticaria; rosacea caused by zoster-associated pain; sarcoidosis;scleroderma; segmental glomerulosclerosis; septic shock syndrome; serumsickness; shoulder tendinitis or bursitis; Sjogren's syndrome; Still'sdisease; stroke-induced brain cell death; Sweet's disease; systemicdermatomyositis; systemic lupus erythematosus; systemic sclerosis;Takayasu's arteritis; temporal arteritis; thyroiditis; toxic epidermalnecrolysis; tuberculosis; type-1 diabetes; ulcerative colitis; uveitis;vasculitis; and Wegener's granulomatosis. The invention also featuresthe treatment of immune disorders that increase susceptibility tomicrobial or viral infection, including HIV.

Diagnostic assays can also be carried out in, e.g., subjects diagnosedor at risk of a proliferative disorder. Such disorders include, withoutlimitation, leukemias (e.g., acute leukemia, acute lymphocytic leukemia,acute myelocytic leukemia, acute myeloblastic leukemia, acutepromyelocytic leukemia, acute myelomonocytic leukemia, acute monocyticleukemia, acute erythroleukemia, chronic leukemia, chronic myelocyticleukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma(Hodgkin's disease, non-Hodgkin's disease), Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors such assarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma,meningioma, melanoma, neuroblastoma, and retinoblastoma).

Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant Pin1 expression or activity. For example, theassays described herein, such as the preceding diagnostic assays or thefollowing assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with Pin1 marker (e.g., animmune disorder or proliferative disorder). Thus, the present inventionprovides a method for identifying a disease or disorder associated withaberrant Pin1 expression or activity in which a test sample is obtainedfrom a subject and Pin1 protein or nucleic acid (e.g., mRNA, genomicDNA) is detected, wherein the presence of Pin1 protein or nucleic acidis diagnostic for a subject having or at risk of developing aPin1-associated disorder and are, therefore, susceptible to treatmentwith a Table 1 Compound.

Furthermore, the present invention provides methods for determiningwhether a subject can be effectively treated with a Table 1 Compound fora disorder associated with aberrant Pin1 expression or activity in whicha test sample is obtained and Pin1 protein or nucleic acid expression oractivity is detected (e.g., wherein the abundance of Pin1 protein ornucleic acid expression or activity is diagnostic for a subject that canbe administered the agent to treat a disorder Pin1-associated disorder).

In one embodiment, the present invention provides methods fordetermining Pin1 post-translational modifications. More importantly,phosphorylation of Pin1 on Ser71 in the catalytic active site may alsoprevent Table 1 Compounds from binding to Pin1 active site and inducePin1 degradation and inhibit Pin1 function. Therefore, detecting reducedSer71 phosphorylation using phospho-specific Pin1 antibodies that wehave generated can be a method to select patients for treatment withTable 1 Compounds and to explain why some patients may not respond totreatment with Table 1 Compounds.

The methods of the invention can also be used to detect geneticalterations in a Pin1 gene, thereby determining if a subject with thealtered gene is at risk for a disorder associated with the Pin1 geneand, consequently, a candidate for therapy with Table 1 Compounds. Inpreferred embodiments, the methods include detecting, in a sample ofcells from the subject, the presence or absence of a genetic alterationcharacterized by at least one alteration affecting the integrity of agene encoding a Pin1-protein, or the mis-expression of the Pin1 gene.For example, such genetic alterations can be detected by ascertainingthe existence of at least one of 1) a deletion of one or morenucleotides from a Pin1 gene; 2) an addition of one or more nucleotidesto a Pin1 gene; 3) a substitution of one or more nucleotides of a Pin1gene; 4) a chromosomal rearrangement of a Pin1 gene; 5) an alteration inthe level of a messenger RNA transcript of a Pin1 gene; 6) aberrantmodification of a Pin1 gene, such as of the methylation pattern of thegenomic DNA; 7) the presence of a non-wild type splicing pattern of amessenger RNA transcript of a Pin1 gene; 8) a non-wild type level of aPin1-protein; 9) allelic loss of a Pin1 gene; and 10) inappropriatepost-translational modification of a Pin1-protein. As described herein,there are a large number of assay techniques known in the art which canbe used for detecting alterations in a Pin1 gene. A preferred biologicalsample is a tissue or serum sample isolated by conventional means from asubject.

In certain embodiments, detection of the alteration involves the use ofa probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the Pin1-gene (seeAbravaya et al. (1995) Nucleic Acids Res 0.23:675-682). This method caninclude the steps of collecting a sample from a patient, isolatingnucleic acid (e.g., genomic DNA, mRNA or both) from the sample,contacting the nucleic acid sample with one or more primers whichspecifically hybridize to a Pin1 gene under conditions such thathybridization and amplification of the Pin1-gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al,(1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a Pin1 gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicate mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531;hereby incorporated by reference) can be used to score for the presenceof specific mutations by development or loss of a ribozyme cleavagesite.

In other embodiments, genetic mutations in Pin1 can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotidesprobes (Cronin, M. T. et al. (1996) Human Mutation 7: 244-255; Kozal, M.J. et al. (1996) Nature Medicine 2: 753-759). For example, geneticmutations in Pin1 can be identified in two dimensional arrays containinglight-generated DNA probes as described in Cronin, M. T. et al. supra.Briefly, a first hybridization array of probes can be used to scanthrough long stretches of DNA in a sample and control to identify basechanges between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the Pin1 gene anddetect mutations by comparing the sequence of the sample Pin1 with thecorresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxam andGilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977)Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any ofa variety of automated sequencing procedures can be utilized whenperforming the diagnostic assays ((1995) Biotechniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT InternationalPublication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr.36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol.38:147-159).

Other methods for detecting mutations in the Pin1 gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the art technique of “mismatch cleavage” startsby providing heteroduplexes formed by hybridizing (labeled) RNA or DNAcontaining the wild-type Pin1 sequence with potentially mutant RNA orDNA obtained from a tissue sample. The double-stranded duplexes aretreated with an agent which cleaves single-stranded regions of theduplex such as which will exist due to basepair mismatches between thecontrol and sample strands. For instance, RNA/DNA duplexes can betreated with RNase and DNA/DNA hybrids treated with S1 nuclease toenzymatically digesting the mismatched regions. In other embodiments,either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine orosmium tetroxide and with piperidine in order to digest mismatchedregions. After digestion of the mismatched regions, the resultingmaterial is then separated by size on denaturing polyacrylamide gels todetermine the site of mutation. See, for example, Cotton et al. (1988)Proc. Nat Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol.217:286-295. In a preferred embodiment, the control DNA or RNA can belabeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in Pin1 cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a Pin1 sequence,e.g., a wild-type Pin1 sequence, is hybridized to a cDNA or other DNAproduct from a test cell(s). The duplex is treated with a DNA mismatchrepair enzyme, and the cleavage products, if any, can be detected fromelectrophoresis protocols or the like. See, for example, U.S. Pat. No.5,459,039; hereby incorporated by reference.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in Pin1 genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci. USA: 86:2766, see also Cotton(1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl9:73-79). Single-stranded DNA fragments of sample and control Pin1nucleic acids will be denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 by of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA86:6230). Such allele specific oligonucleotides are hybridized to PCRamplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner et al. (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a Pin1 gene.

Furthermore, any cell type or tissue in which Pin1 is expressed may beutilized in the prognostic assays described herein.

As with the diagnostic assay described above, prognostic assays of Pin1activity can be included as part of a panel of target genes.

Additional methods of detecting Pin1 activity and diagnosing Pin1related disorders are disclosed in U.S. Patent Application PublicationNos.: 2009/0258352, 2008/0214470, 2006/0074222, 2005/0239095,US2002/0025521, U.S. Pat. No. 6,495,376, and PCT Application PublicationNo. WO02/065091, each of which is hereby incorporated by reference inits entirety.

Monitoring the Effects of Treatment

In one embodiment, the present invention features a method formonitoring the effectiveness of treatment of a subject with a Table 1Compound comprising the steps of (i) obtaining a pre-administrationsample from a subject prior to administration of the compound; (ii)detecting the level of expression or activity of a Pin1 protein, Pin1phosphorylation on Ser71, mRNA, genomic DNA, or other Pin1 marker in thepre-administration sample; (iii) obtaining one or morepost-administration samples from the subject; (iv) detecting the levelof expression or activity of the Pin1 protein, mRNA, or genomic DNA inthe post-administration samples; (v) comparing the level of expressionor activity of the Pin1 protein, mRNA, or genomic DNA in thepre-administration sample with the Pin1 protein, mRNA, genomic DNA, orother Pin1 Marker in the post administration sample or samples; and (vi)altering the administration of the Table 1 Compound to the subjectaccordingly. According to such an embodiment, Pin1 expression,phosphorylation or activity may be used as an indicator of theeffectiveness of the Table 1 Compound, even in the absence of anobservable response.

IV. FORMULATIONS

The inhibitors of the present invention can be formulated intocompositions with an effective amount of the Pin1 inhibitor as an activeingredient. Such compositions can also comprise a pharmaceuticallyacceptable carrier, and are referred to herein as pharmaceuticalcompositions. The inhibitor compositions of the present invention can beadministered intraveneously, parenterally, orally, by inhalation or bysuppository. Formulations of the present invention include thosesuitable for oral, nasal, topical, transdermal, buccal, sublingual,rectal, vaginal and/or parenteral administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. The amount of activeingredient that can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound thatproduces a therapeutic effect. Generally, out of one hundred percent,this amount will range from about 1 percent to about 99 percent ofactive ingredient, preferably from about 5 percent to about 70 percent,most preferably from about 10 percent to about 30 percent. The inhibitorcomposition may be administered in a single dose or in more than onedose over a period of time to achieve a level of inhibitor which issufficient to confer the desired effect.

Suitable pharmaceutical carriers include, but are not limited to water,salt solutions, alcohols, polyethylene glycols, gelatin, carbohydratessuch as lactose, amylose or starch, magnesium stearate, talc, silicicacid, viscous paraffin, fatty acid esters, hydroxymethylcellulose,polyvinyl pyrolidone, etc. The pharmaceutical preparations can besterilized, mixed with auxiliary agents, preservatives, stabilizers,wetting agents, emulsifiers or lubricants such as sodium lauryl sulfateand magnesium stearate, salts for influencing osmotic pressure, buffers,coloring, and/or aromatic substances and the like which do notdeleteriously react with the active compounds. They can also be combinedwhere desired with other active agents, e.g., enzyme inhibitors, toreduce metabolic degradation. Release agents, coating agents,sweetening, flavoring and perfuming agents, preservatives andantioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, .alpha.-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as, for example,cetyl alcohol and glycerol monostearate; absorbents, such as kaolin andbentonite clay; lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and coloring agents. In the case of capsules, tabletsand pills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions that can bedissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions that can be used include polymeric substances andwaxes. The active ingredient can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluent commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants that may berequired.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the activecompound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They may be given by formssuitable for each administration route. For example, they may beadministered in tablet or capsule form, injection, inhalation, eyelotion, ointment, suppository, infusion; topical lotion or ointment; orrectal suppository.

Parenteral administration includes modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion. For parenteral application, injectable, sterilesolutions, oily or aqueous solutions, as well as suspensions, emulsions,or implants, including suppositories, are suitable. Ampoules areconvenient unit dosages.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

It will be appreciated that the actual effective amounts of theinhibitor in a specific case will vary according to the specificcompound being utilized, the particular composition formulated, the modeof administration and the age, weight and condition of the patient, forexample. Dosages for a particular patient can be determined by one ofordinary skill in the art using conventional considerations, (e.g., bymeans of an appropriate, conventional pharmacological protocol).

The regimen of administration can affect what constitutes an effectiveamount. The Pin1 binding compound can be administered to the subjecteither prior to or after the onset of a Pin1 associated state. Further,several divided dosages, as well as staggered dosages, can beadministered daily or sequentially, or the dose can be continuouslyinfused, or can be a bolus injection. Further, the dosages of the Pin1binding compound(s) can be proportionally increased or decreased asindicated by the exigencies of the therapeutic or prophylacticsituation.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient. If desired, theeffective daily dose of the active compound may be administered as two,three, four, five, six or more sub-doses administered separately atappropriate intervals throughout the day, optionally, in unit dosageforms.

V. METHODS OF TREATMENT

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) orafflicted with a disorder associated with increased Pin1 expression oractivity with a Table 1 Compound.

Prophylactic Methods

In one aspect, the invention provides a method for preventing an immunedisorder or proliferative disorder in a subject by administering to thesubject a Table 1 compound. Subjects at risk for a disease which iscaused or contributed to by aberrant Pin1 expression or activity can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a Table 1Compound can occur prior to the manifestation of symptoms characteristicof the Pin1 aberrancy, such that a disease or disorder is preventedand/or its progression delayed.

Combination Therapies

Anti-inflammatory agents are useful for treating an immune disorder orproliferative disorder in combination with the Table 1 Compounds of theinvention. Anti-inflammatory agents that can be used in combination witha Table 1 Compound include, without limitation, corticosteroids, NSAIDs(e.g., naproxen sodium, diclofenac sodium, diclofenac potassium,aspirin, sulindac, diflunisal, piroxicam, indomethacin, ibuprofen,nabumetone, choline magnesium trisalicylate, sodium salicylate,salicylsalicylic acid (salsalate), fenoprofen, flurbiprofen, ketoprofen,meclofenamate sodium, meloxicam, oxaprozin, sulindac, and tolmetin),COX-2 inhibitors (e.g., rofecoxib, celecoxib, valdecoxib, andlumiracoxib), biologics (e.g., inflixamab, adelimumab, etanercept,CDP-870, rituximab, and atlizumab), small molecule immunomodulators(e.g., VX 702, SCIO 469, doramapimod, RO 30201195, SCIO 323, DPC 333,pranalcasan, mycophenolate, and merimepodib), non-steroidalimmunophilin-dependent immunosuppressants (e.g., cyclosporine,tacrolimus, pimecrolimus, and ISAtx247), 5-amino salicylic acid (e.g.,mesalamine, sulfasalazine, balsalazide disodium, and olsalazine sodium),DMARDs (e.g., methotrexate, leflunomide, minocycline, auranofin, goldsodium thiomalate, aurothioglucose, and azathioprine),hydroxychloroquine sulfate, and penicillamine.

In cases where there is a viral or microbial infection, the Table 1Compounds of the invention can be administered with an antimicrobialagent, e.g., the penicillins (e.g., penicillin G, ampicillin,methicillin, oxacillin, and amoxicillin), the cephalosporins (e.g.,cefadroxil, ceforanid, cefotaxime, and ceftriaxone), the tetracyclines(e.g., doxycycline, minocycline, and tetracycline), the aminoglycosides(e.g., amikacin, gentamycin, kanamycin, neomycin, streptomycin, andtobramycin), the macrolides (e.g., azithromycin, clarithromycin, anderythromycin), the fluoroquinolones (e.g., ciprofloxacin, lomefloxacin,and norfloxacin), and other antibiotics including chloramphenicol,clindamycin, cycloserine, isoniazid, rifampin, and vancomycin.Particularly useful formulations contain aminoglycosides, including forexample amikacin, gentamicin, kanamycin, neomycin, netilmicin,paromomycin, streptomycin, and tobramycin, or an antiviral agent, e.g.,1-D-ribofuranosyl-1,2,4-triazole-3 carboxamide, 9→2-hydroxy-ethoxymethylguanine, adamantanamine, 5-iodo-2′-deoxyuridine,trifluorothymidine, interferon, adenine arabinoside, proteaseinhibitors, thymidine kinase inhibitors, sugar or glycoprotein synthesisinhibitors, structural protein synthesis inhibitors, attachment andadsorption inhibitors, and nucleoside analogues such as acyclovir,penciclovir, valacyclovir, and ganciclovir.

Anti-cancer agents that can be used in combination with a Table 1Compound include, without limitation,MK-2206, ON 013105, RTA 402, BI2536, Sorafenib, ISIS-STAT3Rx, a microtubule inhibitor, a topoisomeraseinhibitor, a platin, an alkylating agent, an anti-metabolite,paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide,5-fluorouracil, carboplatin, altretamine, aminoglutethimide, amsacrine,anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil,2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide,cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel,estramustine phosphate, floxuridine, fludarabine, gentuzumab,hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon,irinotecan, lomustine, mechlorethamine, melphalen, 6-mercaptopurine,methotrexate, mitomycin, mitotane, mitoxantrone, pentostatin,procarbazine, rituximab, streptozocin, tamoxifen, temozolomide,teniposide, 6-thioguanine, topotecan, trastuzumab, vincristine,vindesine, and/or vinorelbine.

Such compounds can act synergistically with a Table 1 Compound.Additionally, co-administration with a Table 1 Compound may result inthe efficacy of the anti-inflammatory compound at lower (and thus safer)doses (e.g., at least 5% less (e.g., at least 10%, 20%, 50%, 80%, 90%,or even 95%) less than when the anti-inflammatory compound isadministered alone.

Therapy according to the invention may be performed alone or inconjunction with another therapy and may be provided at home, thedoctor's office, a clinic, a hospital's outpatient department, or ahospital. Treatment optionally begins at a hospital so that the doctorcan observe the therapy's effects closely and make any adjustments thatare needed, or it may begin on an outpatient basis. The duration of thetherapy depends on the type of disease or disorder being treated, theage and condition of the patient, the stage and type of the patient'sdisease, and how the patient responds to the treatment. Additionally, aperson having a greater risk of developing an immune disease orproliferative disorder, may receive treatment to inhibit or delay theonset of symptoms.

Routes of administration for the various embodiments include, but arenot limited to, topical, transdermal, nasal, and systemic administration(such as, intravenous, intramuscular, subcutaneous, inhalation, rectal,buccal, vaginal, intraperitoneal, intraarticular, ophthalmic, otic, ororal administration). As used herein, “systemic administration” refersto all nondermal routes of administration, and specifically excludestopical and transdermal routes of administration.

In combination therapy (e.g., a Table 1 Compound with a secondtherapeutic agent), the dosage and frequency of administration of eachcomponent of the combination can be controlled independently. Forexample, one compound may be administered three times per day, while thesecond compound may be administered once per day. Combination therapymay be given in on-and-off cycles that include rest periods so that thepatient's body has a chance to recover from any as yet unforeseen sideeffects. The compounds may also be formulated together such that oneadministration delivers both compounds.

Each compound of the combination may be formulated in a variety of waysthat are known in the art. For example, the first and second agents maybe formulated together or separately. Desirably, the first and secondagents are formulated together for the simultaneous or near simultaneousadministration of the agents. Such co-formulated compositions caninclude the two drugs together in the same pill, ointment, cream, foam,capsule, liquid, etc. It is to be understood that, when referring to theformulation of combinations of the invention, the formulation technologyemployed is also useful for the formulation of the individual agents ofthe combination, as well as other combinations of the invention. Byusing different formulation strategies for different agents, thepharmacokinetic profiles for each agent can be suitably matched.

The individually or separately formulated agents can be packagedtogether as a kit. Non-limiting examples include kits that contain,e.g., two pills, a pill and a powder, a suppository and a liquid in avial, two topical creams, ointments, foams etc. The kit can includeoptional components that aid in the administration of the unit dose topatients, such as vials for reconstituting powder forms, syringes forinjection, customized IV delivery systems, inhalers, etc. Additionally,the unit dose kit can contain instructions for preparation andadministration of the compositions. The kit may be manufactured as asingle use unit dose for one patient, multiple uses for a particularpatient (at a constant dose or in which the individual compounds mayvary in potency as therapy progresses); or the kit may contain multipledoses suitable for administration to multiple patients (“bulkpackaging”). The kit components may be assembled in cartons, blisterpacks, bottles, tubes, and the like.

VI. PHARMACOGENOMICS

The Pin1 inhibitors described herein can be administered to individualsto treat (prophylactically or therapeutically) disorders (e.g,proliferative disorders such as cancer) associated with aberrant Pin1activity. In conjunction with such treatment, pharmacogenomics (i.e.,the study of the relationship between an individual's genotype and thatindividual's response to a foreign compound or drug) may be considered.Differences in metabolism of therapeutics can lead to severe toxicity ortherapeutic failure by altering the relation between dose and bloodconcentration of the pharmacologically active drug. Thus, a physician orclinician may consider applying knowledge obtained in relevantpharmacogenomics studies in determining whether to administer a Pin1inhibitor as well as tailoring the dosage and/or therapeutic regimen oftreatment with a Pin1 molecule or Pin1 modulator.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons (see, for example, Eichelbaum, M. et al.(1996) Clin. Exp. Pharmacol. Physiol. 23(10-11): 983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254-266). In general, two types ofpharmacogenetic conditions can be differentiated: Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome). Such a high-resolution genetic map can be compared to a map ofthe genome of each of a statistically significant number of patientstaking part in a Phase II/III drug trial to identify markers associatedwith a particular observed drug response or side effect. Alternatively,such a high resolution map can be generated from a combination of someten-million known single nucleotide polymorphisms (SNPs) in the humangenome. Given a genetic map based on the occurrence of such SNPs,individuals can be grouped into genetic categories depending on aparticular pattern of SNPs in their individual genome. In such a manner,treatment regimens can be tailored to groups of genetically similarindividuals, taking into account traits that may be common among suchgenetically similar individuals.

Alternatively, a method termed the “candidate gene approach,” can beutilized to identify genes that predict a drug response. According tothis method, if a gene that encodes a drug target is known (e.g., a Pin1inhibitor of the present invention), all common variants of that genecan be fairly easily identified in the population and it can bedetermined if having one version of the gene versus another isassociated with a particular drug response.

As an illustration, the activity of drug metabolizing enzymes is a majordeterminant of both the intensity and duration of drug action. Thediscovery of genetic polymorphisms of drug metabolizing enzymes (e.g.,N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 andCYP2C19) has provided an explanation as to why some patients do notobtain the expected drug effects or show exaggerated drug response andserious toxicity after taking the standard and safe dose of a drug.These polymorphisms are expressed in two phenotypes in the population,the extensive metabolizer (EM) and poor metabolizer (PM). The prevalenceof PM is different among different populations. For example, the genecoding for CYP2D6 is highly polymorphic and several mutations have beenidentified in PM, which all lead to the absence of functional CYP2D6.Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experienceexaggerated drug response and side effects when they receive standarddoses. If a metabolite is the active therapeutic moiety, PM show notherapeutic response, as demonstrated for the analgesic effect ofcodeine mediated by its CYP2D6-formed metabolite morphine. The otherextreme are the so called ultra-rapid metabolizers who do not respond tostandard doses. Recently, the molecular basis of ultra-rapid metabolismhas been identified to be due to CYP2D6 gene amplification.

Alternatively, a method termed the “gene expression profiling,” can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., a Pin1 inhibitorof the present invention) can give an indication of whether genepathways related to toxicity have been turned on.

Information generated from one or more of the above pharmacogenomicsapproaches, or other approaches known in the art, can be used todetermine appropriate dosage and treatment regimens for prophylactic ortherapeutic treatment an individual. Pharmacogenomic findings, whenapplied to dosing or drug selection, can avoid adverse reactions ortherapeutic failure and thus enhance therapeutic or prophylacticefficiency when treating a subject with a Pin1 inhibitor, such as amodulator identified by one of the exemplary screening assays describedherein.

VII. FURTHER APPLICATIONS OF PIN1 INHIBITORS

The inhibitors of the present invention can be used to interfere witheukaryotic cell growth. The inhibitors can be used in vitro to studycell cycle regulation, mitotic events, protein degradation, apoptosis,neurodegenerative diseases, or certain aspects of cell division such asembryonic development, or signaling pathways thereof. The inhibitors canbe used in defining pathways which lead to carcinogenesis or toevaluate, interfere with, or treat events such as cell spreading inmetastatic cancers. The inhibitors can be used to inhibit cell growth,and to kill targeted cells. For example, the inhibitors of the presentinvention can be used to interfere with the growth of fungus or yeast,including Aspergillus, and parasitic infections (e.g., malaria) inmammals such as domesticated animals and humans.

VIII. EXPERIMENTAL RESULTS Example 1 Pin1 Inhibitor Assay

An assay was developed for the identification of inhibitors of Pin1. Thegoal of this assay was to identify small-molecule inhibitors of Pin1using a fluorescence polarization competition assay (FIG. 1). In thisassay, fluorescence polarization is used to determine whether a givencandidate small molecule inhibitory compound displaces aHiFluor-488-labeled peptide that normally binds to the active site ofPin1. Candidate compounds were screened in a concentration-titrationseries (e.g., 57 μM to 0.7 nM).

Pin1 (0.6 μM) was dispensed into black, solid 1536-well plates (32 rowsby 48 columns) at 2 μL/well in Pin1 buffer (10 mM HEPES (pH 7.5), 10 mMNaCl, 0.01% Tween20, 1 mM DTT, and 1% glycerol). Next, 23 nL of compoundor DMSO was delivered to each well using a pin tool (tip wash sequence:DMSO, IPA, MeOH, 3 s vacuum dry). Two μL of Pin1-1b-HiFluor488 peptidein buffer (10 mM HEPES (pH 7.5), 10 mM NaCl, 0.01% Tween20, 1 mM DTT,and 1% glycerol, 0.02 μM Pin1-1b-HiFluor488) or DMSO was then dispensedto each well using a pin tool (tip wash sequence: DMSO, IPA, MeOH, 3 svacuum dry), and plates were incubated at room temperature for 10 min(Table 2). Each plate included controls with no candidate inhibitorycompound (32 wells), with unlabeled Pin1-lc titration (3.5 mM to 0.020mM in a 1:2 dilution series including 2 replicates per concentration; 32wells), and without Pin1 (64 wells).

TABLE 2 Pin1 Assay Protocol Step Parameter Value Description 1 Reagent 2 μL Pin1 buffer (0.6 μM Pin1; 0 μM controls) 2 Library compounds 23 nLDilution series (10 mM, 7-pt) 3 Control compounds 23 nL DMSO, Pin1-1cdilution series (3.5 mM, 16-pt) 4 Reagent  2 μL Probe 488 buffer 5 Time10 min Room temperature incubation 6 Detection Polarization ViewLux ®

The fluorescence polarization signal of each well was measured on aViewLux® plate reader using a 480/20 nm excitation filter and 540/25 nmS and P emission filters with a 20 second exposure with 2× binning(Table 2). Percent inhibitory activity was determined from correctedfluorescence polarization values. Unlabeled Pin1-1c peptide was used asa control (3.5 mM two-fold serial; 20 μM final concentration) with a1×(0.3 μM) and 0×(no-enzyme) Pin1 enzyme control to normalize thepercent activity of identified inhibitors; Ox enzyme values correspondedto full inhibition, while 1×Pin1 enzyme values were used to normalize noinhibition.

Concentration-response curves were fitted to the signals arising fromthe resulting fluorescence polarization. The concentration-effect curveswere then classified based on curve quality (r²), response magnitude,and degree of measured activity. Compounds were subsequently categorizedbased on their curve class. Active inhibitors showedconcentration-dependent decreases in fluorescence polarization,concordant with their displacement of Pin1-1b-HiFluor488 from the Pin1active site (see, e.g., FIGS. 3A-3D). Inactive compounds showed noeffect on the fluorescence polarization signal.

Example 2 Quantitative High Throughput Analysis of Candidate Pin1Inhibitors

The Pin1 fluorescence polarization competition assay of Example 1 wasused to identify inhibitors of Pin1 from a library of 393,181 compounds(Table 3). Initial analysis was accomplished in an offline LOPAC assay.Ten plates were screened, inclusive of 1,280 compounds, collecting 8,960data points. A hit rate of 1.8% was achieved. A subsequent online LOPACanalysis of 30 plates was also inclusive of 1,280 compounds, collecting26,880 data points. A hit rate of 1.5% was achieved. qHTS of 1607 platesinclusive of 393,181 compounds was then executed, collecting 2,359,086data points. A hit rate of 0.6% was achieved (Table 3). Performance ofthe assay may be gauged using S:B ratio by plate, Z′ score by plate, orCV % by plate (FIG. 2).

TABLE 3 Pin-1 Inhibitor Screens and overall Plate Statistics ParameterOffline LOPAC Online LOPAC qHTS System Offline Kalypsys robot Kalypsysrobot Plates screened 10 30 1607 Plates failed QC 0 0 2 (DMSO plate)Compounds tested 1,280 1,280 393,181 Concentration- 3 nM - 57 μM 3 nM -57 μM 3 nM - 114 μM Response Titrations No. of data points 8,960 26,8802,359,086 Z′ 0.79 +/− 0.03 0.69 +/− 0.04 0.72 +/− 0.04 Signal/Background3.29 +/− 0.11 2.48 +/− 0.09 2.57 +/− 0.09 CV  5.2 +/− 3.45  6.9 +/− 1.935.6 +/− 2.3 MSR (Pin1-1c) 1.3 1.6 1.2 Hit rate (high-quality 1.8% 1.5%0.6% (2,315) actives)

Example 3 Identification of Pin1 Inhibitors

Data collected from the Pin1 fluorescence polarization competitionassays of Example 2 may be expressed as curves showing log concentrationof compound (X axis) by change polarized fluorescence or totalfluorescence (FIGS. 3A-3E). Curves showing change polarized fluorescencemay be categorized into 5 classes. Class 1 includes complete curves,having 2 asymptotes and an r² value of ≧0.9, with an efficacy ofeither >80% (class 1.1) or <80% (class 1.2). Class 1 curves may also beclassified as noisy curves when efficacy is >80% and r²<0.9 (class 1.3)or when efficacy is <80% and r²<0.9 (class 1.4). Class 2 includesincomplete curves having 1 asymptote and an r² value of either >0.9(subclass a) or <0.9 (subclass b), with an efficacy of either >80%(class 2.1) or <80% (class 2.2). Class 2 curves may also be classifiedas noisy curves when efficacy is >80% and r²<0.9 (class 2.3) or whenefficacy is <80% and r²<0.9 (class 2.4). Class 3 includes single pointactivity curves having 1 asymptote and an efficacy >3 SD from the meanactivity of the sample field at the highest tested concentration. Class4 includes inactive curves, for which there are no asymptotes and forwhich efficacy and r² value are not applicable. A fifth class capturesany curves not otherwise classified (FIG. 4).

Analysis of results from the Pin1 fluorescence polarization competitionassays of Example 2 reveal that 99% of compounds (388,068 compounds)yield data curves classified as inactive. Active inhibitors includecompounds yielding class 1 and class 2 curves and efficacy greater thanor equal to 50%. These represented 0.6% of tested compounds, a total of2315 compounds. Of these, 971 (42%) demonstrate no interference of totalfluorescence, 114 (5%) may demonstrate interference of totalfluorescence, 1117(48%) demonstrate strong interference of totalfluorescence, and 113 (5%) demonstrate a possibility that they quenchtotal fluorescence (FIG. 5). Excluding those that demonstrate stronginterference of total fluorescence, 1198 compounds were selected forfurther analysis. Exemplary Pin1 inhibitors, their chemical structures,and associated data are shown in Tables 1 and 4.

Example 4 Secondary Assessment of Pin1 Inhibitors

To further evaluate the Pin1 inhibitors identified in the Pin1fluorescence polarization competition assays of Example 2, as analyzedin Example 3, 1086 of the 1198 identified inhibitors of Pin1 wereretested in a confirmatory Pin1 fluorescence polarization competitionassay. Of the 1086 compounds, 307 yielded class 1.1, 1.2, 2.1, or 2.2curves and efficacies greater than 50%.

A further secondary assessment was accomplished by replacing theHiFluor-488-labeled probe with Tamra-probe, a red-shifted fluorophore.In the Tamra assay, 285 of the 1086 compounds yielded class 1.1, 1.2,2.1, or 2.2 curves and efficacies greater than 50%.

Of the compounds evaluated in both secondary assessments, 220 compoundsyielded class 1.1, 1.2, 2.1, or 2.2 curves and efficacies greater thanor equal to 50% in both. In total 191 compounds met these criteria inall three assays.

TABLE 4 Compound numbers correspond to Table 1 Compounds Average AverageHTS HTS Alexa488 Primary Alexa488 Reconfirmed Tamra Secondary PotencyEfficacy Potency Efficacy Curve Potency Efficacy Curve Potency EfficacyCurve • (μM) (% Inh.) (μM) (% Inh.) Class (μM) (% Inh.) Class (μM) (%Inh.) Class 1 3 84 6 66 1.2 1.30 90 1.1 1.20 95 1.1 2 4 81 7 86 1.1 2 891.1 3 69 1.2 3 6 71 10 80 1.1 3 61 1.2 5 72 1.2 4 7 30 3 23 1.2 nr nr 1137 2.2 5 7 63 18 53 1.2 2 75 1.2 2 61 1.2 6 14 72 22 42 1.1 10 85 11 872.1 7 29 72 45 63 1.2 30 91 2.2 14 63 2.2 8 31 74 45 67 2.2 30 103 2.218 52 2.2 9 4 95 10 78 1.3 2 111 1.1 1.20 97 1.1 10 13.6 82.1 22.4 81.32.2 4.8 82.9 1.1 11 3 83 3 65 1.2 3 93 1.1 4 91 1.1 12 6 88 15 78 1.21.20 93 1.1 1.10 92 1.1 13 4 89 9 89 1.1 2 89 1.1 3 88 1.1 14 10 72 2171 1.2 1 59 1.2 8 85 2.1 15 11 133 10 27 2.2 11 71 2.2 12 300 2.1 16 2777 76 54 1.1 2 93 1.1 2 84 1.1 17 5 79 10 64 1.4 2 83 1.1 3 89 1.1 18 2477 68 44 1.1 2 91 1.1 2 95 1.1 19 15 64 38 41 1.2 2 73 1.2 5 78 2.2 20 773 14 73 1.1 2 77 1.2 6 70 1.2 21 11 62 15 58 1.2 11 56 2.2 8 72 2.2 2217 88 n/a n/a n/a 21 93 2.2 12 82 2.2 23 33 77 76 63 2.4 15 92 2.1 8 772.2 24 23 92 43 76 1.2 14 101 1.2 12 100 2.1 25 34 47 38 22 2.2 nr nr 3073 3.0 26 22 94 20 71 1.1 24 111 2.1 21 101 2.1 27 27 108 23 85 1.1 30112 2.1 27 126 2.1 28 0.89 94 1.69 89 1.1 0.08 99 1.1 29 0.90 85 3 831.1 0.10 90 1.1 0.10 82 1.1 30 1.16 79 3 69 1.2 0.34 89 1.1 0.43 80 1.131 2 80 4 74 1.1 2 85 1.1 2 82 1.1 32 13 61 38 21 1.2 0.24 82 1.1 0.1981 1.1 33 21 58 48 20 1.2 7 84 2.2 10 70 2.2 34 27 91 20 65 1.4 24 1042.1 38 103 3.0 35 19 71 38 26 2.4 9 112 2.1 11 75 2.2 36 15 80 11 1011.1 14 62 2.2 19 76 2.2 37 37 47 85 19 1.2 15 63 2.2 12 58 2.2 38 13 10119 103 1.1 6 98 1.1 39 17 41 14 28 2.2 nr nr 21 53 3.0 40 20 83 25 551.1 10 99 2.1 24 96 2.2 41 23 77 30 88 2.2 19 62 2.2 19 80 2.2 42 23 7619 73 1.2 27 82 2.2 24 73 2.2 43 20 67 16 63 1.2 nr nr 24 71 3.0 44 2479 36 90 2.2 12 63 2.2 24 83 2.2 45 30 71 22 72 2.1 nr nr 38 71 3.0 4630 81 43 82 2.2 27 84 2.2 21 78 2.2 47 40 67 79 26 1.2 19 84 2.2 21 912.1 48 1 63 1 53 1.4 1 73 1.1 49 25 48 50 27 1.2 1 70 1.1 50 4 95 1 931.1 7 98 1.1

Example 5 A Pilot Screen for Pin1 Inhibitors Identified anAnti-Proliferative Agent

A high-throughput screen for identifying Pin1 inhibitors was developedusing a single-step fluorescence polarization-based displacement assay(FP-HTS). The FP-HTS detects molecules that compete for the substratebinding to the catalytic active site, measures ligand binding underequilibrium conditions, and does not suffer from product inhibition. TheHF488 fluorescent probes for the FP assay contain only four residue corestructure of Bth-L-phos.Thr-Pip-Nal (pTide), with a Kd of 258 nM forPin1, was synthesized by Anaspec. We performed FP-HTS in a 384-wellplate format with full length Pin1 and produced robust FP, resulting ina 6-7 fold increase in polarization degree values, using a Synergy IIplate reader. This novel FP-HTS showed robust and reproducibleperformance. The assay can tolerate up to 10% DMSO. The Z′ is around0.70 and is consistent for day-to-day performance. The coefficient ofvariation is in the range of 4-5%. More importantly, we have shown thatboth pTide, the unlabeled Pin1 peptide as the positive control in thisproject, and juglone, the Pin1 small molecule inhibitor, displaced HF488probe from Pin1. Although it is difficult to determine the Kd for thecovalent and irreversible inhibitor juglone, the Kd for pTide was −250nM, similar to that derived from PPIase-based assays.

The FP-HTS with a 5 nM probe and 200 nM Pin1 was used to conduct a pilotscreen on a selected set of chemical libraries. We obtained theresulting potential positive hits and grouped them into 3 classesaccording to the Z-score, which is folds of standard deviation below themean. The top inhibitory chemical was the clinically used drug13-cis-retinoic acid (cis-RA). Characteristics of cis-RA is includethat 1) cis-RA is not listed in promiscuous inhibitor databases 2) thepairing compound of cis-RA, all-trans retinoic acid (trans-RA), iscurrently used as oral prescription for patients with acutepromyelocytic leukemia (AML); 3) both cis-RA and trans-RAs are used as amedication in the phase 11/111 clinical trial for breast cancer, andmore because of its ability to kill rapidly dividing cells; and 4)although it has been reported that RAs target on retinoic acid receptor(RAR), the exact mechanism of the anticancer action is unknown.

The anti-cancer effect of RAs was examined to determine whether itdepends on RAR in the breast cancer cells. RARa knockdown can onlypartially rescue-cis-RA-mediated cell death, indicating that RAs mayhave unidentified “off-target effect.” To confirm that RAs indeed targetPin1, we examined cis-RA and trans-RA in the FP assay, and,surprisingly, found that trans-RA displayed even more prominent Pin1inhibition than cis-RA and that cis-RA would eventually catch up withtrans-RA in the long-term incubation with Pin1, likely due toresonance-mediated cis-trans conversion. In the PPIase assay, Pin1activity was blocked by either cis- or trans-RA in a dose-dependentmanner. These data confirm that the interaction between RAs and Pin1 isspecific and not due to aggregation. Furthermore, both trans and cis RAblocked the association between Pin1 and DAPK1 in a dose-dependentmanner, with trans being more potent. These results indicate that RAsbinds to Pin1 C-terminal catalytic domain because DAPK1 is known to bindthis domain (Lee et al., 2011 Mol Cell in press). In determining whichamino acid residues in the Pin1 catalytic domain are important forretinoic acid binding, point mutations of Pin1 including K63A, S67E,R68/69A, H59A or S71E completely or significantly abolished trans-RAbinding to Pin1. Together, these data indicate that RAs inhibit Pin1 byoccupying its catalytic PPIase pocket in the C-terminus and thatphosphorylation of Ser67 or Ser71 inhibits RA binding to Pin1.

To further discern the causal relationship between theanti-proliferative effect of RAs on Pin1, cell viability of three breastcancer cell lines was tested with different dosages of cis- or trans-RA,of which SKBR3 and T47D exhibited preferential sensitivity to RAs withan IC50 in the nano-molar range, while the normal cell line MCF10Aremained unaffected. This discrepancy between cell lines was correlatedwith the RAs' ability to suppress Pin1 expression. Pin1 level wasdecreased by treatment of RAs in drugs-responsive SKBR3 and T47D, butnot in drugs-irresponsive normal cells, MCF10A, in which Pin1 targetprotein, cyclin D1, served as biomarker of in vivo Pin1 activity.

Moreover, RAs did not alter Pin1 mRNA level, but did reduce Pin1 proteinstability, suggesting that RAs interact with Pin1, result in Pin1degradation, and subsequently leads to anti-proliferation of breastcancer cells. To further confirm this premise, wild-type mouse embryonicfibroblast (WT MEF) and Pin1 knockout MEF (Pin1 KO MEF) were used totest trans-RA-mediated cell viability. As expected, Pin1 KO MEF was moreresistant to trans-RA than WT MEF due to lack of drug target. Inaddition, Pin1 KO MEF stably expressing WT Pin1, but not W34 K63A Pin1mutant, enabled cells to re-sensitize trans-RA. These results indicatethat RAs-mediated cell death is at least in part dependent on Pin1.

We have further shown that combinations of therapeutic compoundsincluding retinoic acid compounds are useful for treating cancer, e.g.,cancer characterized by elevated Pin1 activity. Results obtained fromthe treatment of breast cancer cells overexpres sing Pin1 with ATRA orDoxorubicin or their combination, followed by counting cancer cellnumbers show that ATRA and Doxorubicin combination dramaticallyincreases anticancer potency and reduce the dose of each drug to inhibitcancer cell growth. Therefore, ATRA can drastically reduce dose andtoxicity of Doxorubicin and other chemotherapeutic drugs.

We have also shown that Pin1 inhibition, using siRNA, dramaticallyreduces Neu/Erb2 overexpression and cell proliferation of human breastcancer cells that have Neu/Erb2 gene amplification. This provides amethod to identify a Pin1 modulatory compound by applying a testcompound to human-derived cancer cells that have Neu/Erb2 geneamplification, and determining the effect of the test compound onNeu/Erb2 overexpression and cell proliferation.

In addition, we have developed cell-based assays to screen and validatePin1 inhibitor hits. We have shown 1) that Pin1 is highly expressed inHER2-positive human breast cancer tissues; 2) that Pin1 inhibitionalmost completely suppresses HER2 overexpression on cell surface inhuman HER2+ breast cancer cell lines such as AU565 and SKBR3 cells; 3)that Pin1 inhibition greatly increases the sensitivity of HER2+ breastcancer cells to the mTOR inhibitor, but not to the HER2 inhibitor,suggesting that Pin1 might act on Her2 to regulate cell growth; 4) thatPin1 acts on Neu and multiple substrates in Neu-mediated oncogenicpathway; and 5) that Pin 1 knockout in mice inhibits breast cancerdevelopment induced by activated Her2. Therefore, Pin1 is essential formaintaining HER2 overexpression and growth of human HER2+ breast cancercells. Given that HER2 expression on cell surface and cell growth arereadily assayed on 384-well format, we can test the ability of the hitsto repress HER2 overexpression and cell growth of HER2_AU565 and SKBR3cells, which will be treated with Pin1 prodrugs or hits, and thenimmunostained with Alexa 488-anti-HER2 monoclonal antibody (BioLegend),followed by automated microscopy.

OTHER EMBODIMENTS

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth, and follows in the scope ofthe claims.

Other embodiments are within the claims.

What is claimed is:
 1. A method of inhibiting Pin1 by contacting Pin1with a compound having the structure selected from Compound 48 andCompound 49:

wherein said compound is a Pin1 inhibitor.
 2. The method of claim 1,wherein said Pin1 is in a cell.
 3. The method of claim 2, wherein saidcell is a human cell.